1. Field of the Invention
This invention relates to a process for producing the protein part of Ca-binding photoprotein aequorin (i.e. apoaequorin) employing a recombinant DNA technique, characterized by recovering the apoaequorin outside the bacterial cells and a process for purifying the apoaequorin from the filtrate of the resulting culture media.
2. Description of the Related Art
Natural aequorin is a photoprotein present in the cells existing on the periphery of the umbrella of photogenic Aequorea growing near the Harber island, Friday, Wash., U.S.A., and only about 200 mg of purified aequorin is obtained from about 5 tons of Aequorea.
Since this photoprotein aequorin is specifically bound with Ca.sup.++ ion to emit light, it has been utilized for various analyses. However, since the quantity fed is insufficient, its availability (in the analysis utilizing a faint light emittance) has not yet been sufficiently effected.
Recently, the present inventors have succeeded in producing a recombinant aequorin having an aequorin activity, by producing the protein part of aequorin (apoaequorin) according to recombinant DNA technique, followed by mixing it with a reducing agent, a substrate, etc. (Japanese patent application Nos. Sho 60-280259/1985 and Sho 61-249098/1986).
However, production of apoaequorin in a large quantity and its purification have not yet been achieved. Thus, we have investigated various culture conditions using an expression vector piP-HE outside the bacterial bodies (Japanese patent application No. Sho 61-249098/1986). As a result, it has become possible to detect production of apoaequorin outside the bacterial cells i.e. in the culture filtrate in a similar quantity (20 .mu.g-50 .mu.g/ml) to that in the bacterial cells.